The goal common to all of our experiments is to learn details of mammalian gene regulation and control. Research topics address the molecular genetics of embryonic development. The determination of LIM homeodomain (LIM-HD) protein function has remained a focus of our studies. The LIM-HD proteins play fundamental roles in development. Their primary structure has been remarkably well preserved during the course of evolution. Functional data have been derived from mutational analysis in such diverse organisms as nematodes, flies and vertebrates. These studies have revealed a prominent involvement of LIM-HD proteins in tissue patterning and differentiation. Their function in neural patterning is evident in all organisms. Our previous work addressed the function of several LIM-HD proteins during the development of the mouse embryo, based on the analysis of knockout mutants. In particular, we analyzed the roles of Lhx3 and Lhx4 in cells giving rise to the pituitary and to motor neurons, of Lhx5 in the developing hippocampus, and of Lhx2 in brain, eye and hemapoietic development. Topics of our current studies are the functions of two other LIM-HD genes, Lhx8 (also known as L3 or Lhx7), and Lhx9.Lhx8, together with Lhx6, belongs to a subgroup of vertebrate LIM-HD genes that are expressed in the first branchial arch and in the forebrain of the developing mouse embryo. Lhx8 transcripts are found in the maxillary and mandibular processes and in the ventral forebrain. We generated an Lhx8 knockout mouse to analyze the function of this gene. Null mutants are characterized by an isolated cleft palate. Development of other craniofacial structures appeared normal. The primordial palatal shelves formed and elevated normally on both sides of the midline, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. This defect is most likely caused by an intrinsic defect in functions that mediate interactions between epithelial and mesenchymal cells required for the fusion of the palatal shelves. The cleft palate phenotype shows incomplete penetrance, suggesting that these functions are the product of an interaction of Lhx8 with other cleft palate susceptibility genes or teratogens. A similar phenotype has previously been observed in mice with targeted null mutations in the genes encoding TGFbeta 3, EGFR and TTF2. The multifactorial origin of craniofacial clefting, a frequent congenital disorder, is well recognized in human genetics. The human homolog of the mouse Lhx8 gene maps to a chromosomal region that has been linked to craniofacial clefting, suggesting a screen for Lhx8 mutations in patients with cleft palate and their kindreds. Lhx9 and its close structural relative Lhx2 are members of the apterous group of LIM-HD genes. Their expression patterns overlap in the developing limbs. In the embryonic brain, there are some overlaps but also distinct regions of expression. Notably, Lhx9 transcripts were detected in the urogenital ridge and later in the gonads of the developing embryo. A function of Lhx9 in gonad formation was revealed by the phenotypic analysis of mice homozygous for a targeted null mutation of the gene. The mutants were viable but infertile. All animals, whether genotypically male or female, lacked gonads and had an atrophic uterus and vagina. Levels of testosterone and estradiol were minimal, those of follicle stimulating hormone were high. In Lhx9-/- mutant embryos, germ cell migration to the genital ridge was unaffected, but cells of the gonadal ridge failed to proliferate, and the definitive gonad did not form. Phenotypes similar to that of Lhx9-/- mutant mice have been observed in certain human congenital disorders, suggesting that this gene might be affected in some forms of human gonadal agenesis. A mouse model for Hirschsprungs disease. Hirschsprungs disease is a frequent human congenital disorder that is caused by defective innervation of the distal gastrointestinal (GI) tract. Hirschsprung patients suffer from chronic constipation and gut obstruction that can be life-threatening. Several gene defects affecting the GDNF/c- Ret signalling pathway have been associated with the human disorder. In earlier reports (see Z01 HD-00071-24 and 25-LMGD), we described a knockout mouse carrying a null mutation in the gene encoding glial cell line derivedneurotrophic factor (GDNF), a ligand ofthe c- Retreceptortyrosine kinase.GDNF is a major neurotrophic factor for enteric neurons, and it promotes ureter branching during kidney development. GDNF homozygous null mutants are characterized by enteric aganglionosis and kidney agenesis and die at birth. By contrast, the phenotype of mice that are heterozygous for the GDNF null mutation reflects important aspects of the c-Ret associated human disease. A transgenic marker strategy enabled us to establish ganglion cell density along the GI tract of normal and GDNF+/- heterozygous mutant animals. The mutant cohort is characterized by a general hypoganglionosis of the GI tract. Defective gut innervation causes symptoms related to gut dysmotility, and up to 20% of the GDNF+/- animals die, most of them shortly after birth. Like in children with Hirschsprungs disease, the inheritance of the mutant phenotype is autosomal dominant, the penetrance incomplete, the expressivity of symptoms variable, and the onset of morbidity and mortality congenital. The study of the GDNF+/- mutant mice has provided valuable insights into general mechanisms of predisposition and susceptibility to Hirschsprungs disease. Sonic Hedgehog. The work outlined here was begun when Drs. Litingtung and Chiang were working as postdoctoral trainees at LMGD and deals with specific functions of sonic hedgehog (Shh), a secreted morphogen that has been extensively studied in this laboratory (see Z01 HD-00071-23 and 25-LMGD). Mice with a targeted deletion of the Shh gene have foregut defects that become apparent at day E9.5 of gestation when the tracheal diverticulum forms. Shh-/- null mutant embryos show a number of defects in esophagus, trachea and lung development, similar to those seen in a frequent human congenital disorder that is caused by malformation of the foregut. We observed decreased cell proliferation, enhanced cell death and down-regulation of Shh target genes in the affected tissues. Shh is thus required for the growth and differentiation of esophagus, trachea and lung, and mutations of Shh or its signalling components may be responsible for the human foregut defects. In a separate study, carried out in collaboration with the laboratories of Drs. Philip Beachy (Johns Hopkins U.) and Andrzej Dlugosz (U. Michigan, Ann Arbor), the role of Shh in hair follicle morphogenesis was investigated. The hair follicle is of major clinical interest as the origin of several types of skin tumors. In Shh-/- null mutants, dermal condensates failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Shh is thus essential for hair follicle development beyond the hair germ stage. Further studies are required to explain how constitutive activation of the Shh signaling pathway in keratinocytes contributes to the formation of basal cell carcinoma.